Enzyme Assays Glucose Oxidase Activity

The major enzymes used in ELISA technology include horseradish peroxidase (HRP), alkaline phosphatase (AP), (3-galactosidase (P-gal), and glucose oxidase (GO). See Chapter 26 for a detailed description of enzyme properties and activities. HRP is by far the most popular enzyme used in antibody-enzyme conjugates. One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody conjugates, most of them utilized in diagnostic assay systems. 

The [Eu(Tc)] assay for H202 can also be used for the determination of enzyme activities. This comprises all enzymes that produce (oxidases) or consume (catalase, peroxidases) H2O2. For example, the activity of glucose oxidase can be directly imaged after the addition of [Eu(Tc)] as indicator and glucose as substrate. Steady-state and time-resolved imaging schemes can be applied for the read-out of a microwell plate-based assay. The best results were obtained by the RLI method (Fig. 19) [115]. 

Polyelectrolyte multilayer microspheres, prepared by alternating adsorption of dextran sulfate and protamine on melamine formaldehyde cores followed by the partial decomposition of the core, were used to immobilise the peroxidase and glucose oxidase. Retention of enzymic activity of the peroxidase/glucose oxidase system incorporated into the microspheres was demonstrated. These bienzyme system immobilised in the microspheres can be applied for kinetic glucose assays [ 156]. 

Calculation One Glucose Oxidase Titrimetric unit of activity (GOTu) is the quantity of enzyme that will oxidize 3 mg of glucose to gluconic acid under the conditions of the assay. Determine the enzyme activity using the following equation ... 

Enzyme Assay Procedure. The catalytic potency of the immobilized g-galactosidase was determined in a plug flow reactor ( 9). Glucose liberated by the catalytic activity of 3-galactosi-dase on lactose was determined by the glucose oxidase-chromogen method (21 ) with some modifications. 

Detection limits in EIA are ultimately determined by how low one can measure the label s concentration via an activity assay. Sensitivity in such a kinetic determination is dependent upon the turnover number of the enzyme molecule and the method employed to detect the product of the catalyzed reaction. Purified urease obtained from Sigma Chemical Co. has considerably higher activity on a molar basis (international units per mole of enzyme) than the best available commercial preparations of some other common enzyme labels such as alkaline phosphatase, /8-galactosi-dase, peroxidase, - and glucose oxidase. This is due to the high mo-... 

Other minor enzyme systems can be used, such as P-galactosidase (5). This system works well. However, it can lead to some false-positive problems resulting from endogenous enzymes which have a similar reactivity (6). Another enzyme not commonly used is glucose oxidase. A glucose oxidase system can provide a sensitive and specific assay if other endogenous enzyme activity is a problem. 

Another example concerns the immobilization of a biocatalyst—the enzyme glucose oxidase (GOD)—to the silica nanotubes [4]. GOD was immobilized, on both the inside and outside surfaces, via the aldehyde silane route. These GOD-nanotubes (60 nm diameter) were dispersed into a solution containing 90 mM glucose and also the components of the standard dianisidine-based assay for GOD activity. A GOD activity of 0.5 0.2 units per milligram of nanotubes was obtained. These studies also showed that protein immobilized via the Schiff base route is not leached from the nanotubes, where GOD activity ceased when the nanotubes were filtered from the solution. 

The direct measurement of analyte concentrations below approx. 10 mol/L with electrochemical sensors is so far only possible with the advanced microfabrication process of Ikarijama et al. [21-23]. Usually a chemical amplifier system has to be used for increased sensitivity. The only analytes which already provide this amplification system themselves are enzymes catalytically turning over their substrates. If the product of the enzymatic reaction can be assayed with a sensor, the activity of the respective enzyme can also be determined. As an example, an amperometric assay for glucose oxidase using benzoquinone as an oxidant has been published [37] ... 

The response of a surface antigen-antibody reaction can also be mediated by an electron transfer reagent. This has been demonstrated by Robinson et al. in their studies on electrochemical immunoassays for hCG and thyroxine. In the analysis of hCG, a two-site amperometric immunoassay was developed in which monoclonal capture antibodies were immobilised on the surface of a glassy carbon electrode. A second antibody against hCG was labelled with glucose oxidase. The electrode was used both to separate free from bound enzyme-antibody conjugate and to assay the enzyme activity electrochemically by use of dimethylaminomethyl ferrocene as an electron transfer mediator. This method was found to correlate well with an immunoradiometric assay. In the analysis of thyroxine, another ferrocene derivative, namely ferrocenemonocarboxylic acid, was used as the electron transfer mediator. ... 

Enzyme-catalysed reactions are widely used for analytical purposes, for the determination of substrates (e.g. glucose oxidase for determination of glucose) and of inhibitors (such as pesticides, by their inhibition of cholinesterase) and activators. Although enzymes are very useful as analytical reagents, they are not classified individually in this Dictionary. However, enzymes themselves are extensively assayed by clinical chemists, biochemists, forensic scientists and food chemists, and the substrates used for such assays are carefully chosen to achieve optimum sensitivity, selectivity and reliability. Such substrates are listed in this Dictionary, as are the co-enzymes (co-factors) required by many redox enzymes, for example nicotinamide adenine dinucleotide (NAD /NADH) which is a co-enzyme for many dehydrogenases, e.g. 

Hydrogen peroxide is produced by the activity of oxidases and determining its intracellular concentration provides assays of the activity of the corresponding enzyme or of substrates such as glucose. It can be detected with high sensitivity by chemiluminescence or electroluminescence, however only at relatively high pH values. A lanthanide-based analytical procedure was proposed in 2002 by O. Wolfbeis and collaborators,... 

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